Antioxidants in the Fight Against Atherosclerosis: Is This a Dead End?
Current Atherosclerosis Reports
Antioxidants in the Fight Against Atherosclerosis: Is This a Dead End?
Paola Toledo-Ibelles 0
Jaime Mas-Oliva 0
0 Instituto de Fisiología Celular, Universidad Nacional Autónoma de México , Mexico City , Mexico
1 Jaime Mas-Oliva
Purpose of Review The purpose of this review is to focus on the outcome of recent antioxidant interventions using synthetic and naturally occurring molecules established as adjuvant strategies to lipid-lowering or anti-inflammatory therapies designed to reduce the risk of cardiovascular disease. Recent Findings To date, accumulated evidence regarding oxidation as a pro-atherogenic factor indicates that redox biochemical events involved in atherogenesis are indeed a very attractive target for the management of cardiovascular disease in the clinic. Nevertheless, although evidence indicates that redox reactions are important in the initiation and progression of atherosclerosis, oxidation with a pro-atherogenic context does not eliminate the fact that oxidation participates in many cases as an essential messenger of important cellular signaling pathways. Therefore, disease management and therapeutic goals require not only highprecision and high-sensitivity methods to detect in plasma very low amounts of reducing and oxidizing molecules but also a much better understanding of the normal processes and metabolic pathways influenced and/or controlled by oxidative stress. As several methodologies have been specifically described for the quantification of the total antioxidant capacity and the oxidation state of diverse biological systems, a successful way to carefully study how redox reactions influence atherosclerosis can be achieved. Summary Since there is still a lack of standardization with many of these methods, clinical trials studying antioxidant capacity have been difficult to compare and therefore difficult to use in order to reach a conclusion. We believe a comprehensive analysis of new knowledge and its relationship with the presence of plasma antioxidants and their reducing capacity will undoubtedly open new ways to understand and develop new therapeutic pathways in the fight not only against atherosclerosis but also against other degenerative diseases. This article is part of the Topical Collection on Nonstatin Drugs
Atherosclerosis; Cardiovascular disease; Antioxidant therapy; Redox reactions; Standardization factors
Oxidative Stress and Atherosclerosis
Cardiovascular diseases (CVDs) are nowadays considered the
clinical complications with the greatest impact on mortality in
the western world. According to WHO’s most recent records,
CVDs were responsible for a total of 46% of deaths due to
noncommunicable diseases in 2012 [
]. Among CVDs, coronary
heart disease and stroke are the ones with the greatest impact
since in addition to causing a high frequency of deaths, they
occur at ages that are still far from the maximum life
expectancy as rated by WHO [
]. These diseases are the clinical
manifestation of the pathophysiological process known as
atherosclerosis, related to inflammation and the accumulation in
the blood vessels of the ketone and hydroxide forms of lipids
derived from the non-enzymatic oxidation of cholesterol and
polyunsaturated fatty acids (PUFA) [
]. It has been proposed
that these lipids originate from low-density lipoproteins
(LDLs), which in their oxidized form (oxLDL) are taken up
by receptors such as LOX-1, SR-A, and SR-B. Unlike the
receptor that recognizes native low-density particles (LDLR),
these are not regulated by intracellular cholesterol levels and
allow an excessive increase of cholesterol inside the cell
generating the so-called macrophage-derived foam cells [
Following pioneer studies carried out by Goldstein and
Brown who first proposed a binding site on macrophages for
chemically modified LDL uptake such as acetylated and
maleylated LDL [
]; in the 1980s and the 1990s, classical
studies by J.L. Witztum defined that an important LDL
modification is associated with the oxidation of LDL particles [
incubating LDL with cultured endothelial cells or smooth
muscle cells, they showed that the newly created oxidized
forms are rapidly internalized in a saturable manner.
Currently, the most widely accepted hypothesis regarding
atherogenesis proposes that atherosclerosis initiates its
development with LDL entrance from the bloodstream into the
subendothelial space, between the tunica intima and media,
where cellular metabolism fosters its oxidation, phagocytosis
by macrophages and their consequent overloading of
intracellular lipids [
]. Moreover, oxLDLs act as a chemotactic
factor for monocytes and induces epigenetic modifications that
exacerbate proinflammatory cytokine production [
Also, PPAR-γ activation by the oxidized lipid fraction leads
to differentiation into macrophages where oxLDL-stimulated
macrophages are prone to migrate through a mechanism
dependent on intracellular nitrosative stress and lipid peroxidation
favoring their accumulation in the plaque [
since modified lipoproteins affect vascular cells as well, it has
been found that endothelial cells increase their level of
intracellular oxidative stress without oxLDL internalization due to the
formation of reactive oxygen species (ROS) that permeate
through the cell membrane . Additionally, proinflammatory
cytokines stimulate the proliferation and migration of smooth
muscle cells, confining foam cells within fibrous tissue through
mechanisms dependent on MAPK and NF-KB signaling [
where a cytokine such as osteopontin involved in inflammatory
and calcification processes promotes atheroma growth [
In this way, ROS and reactive nitrogen species (RNS) present
in the vascular environment create a pro-atherogenic condition
exacerbated by the presence of an impaired equilibrium
between the oxidizing and reducing capacity of the cell, a state
known as oxidative stress.
Thus, the importance of oxidative stress in the development
of atherosclerosis also relies on cellular responses triggered by
an inadequate equilibrium between oxidants and reductants at
different layers of the vascular tissue . The diverse
oxidation-reduction reactions that take place in this tissue
consist of a strict exchange of electrons between molecules
generating specific electric potentials (E°′) that can be modified by
diverse tissue conditions. The electric potential will be greater
for compounds prone to be reduced, where the most favorable
reaction occurs between the strongest oxidant and the strongest
reductant present in the reaction mixture , mostly mediated
by ROS and RNS . Although ROS and RNS present the
capacity to oxidize many biomolecules, reductant molecules
present in the microenvironment present the property to
counteract the process. A strong oxidant such as the hydroxyl-radical
might react with a strong reductant such as ascorbic acid,
preventing an unwanted oxidation process and therefore
becoming an “antioxidant,” a bioavailable reductant molecule that
could prevent the progress of oxidative stress .
While the difference in potential provides information
about the physicochemical characteristics of the reaction, it
is still dependent on the concentration of reactants as well as
on cellular location. Although ascorbic acid (E°′ = 282 mV) is
a stronger reductant than α-tocopherol (E°′ = 500 mV), the
hydrophobic nature of tocopherol increases its antioxidant
capacity, for instance against LDL-lipoperoxide formation,
reason why tocopherol has attracted so much attention as an
antioxidant in clinical research . Since oxidation of lipids
in plasma only occurs when ascorbic acid and α-tocopherol
are, in turn, also oxidized , in a biological context, redox
reactions depend on factors such as affinity of these molecules
for lipoproteins, membranes, and/or diverse cellular
compartments  (Fig. 1). Although the clinical approach for the use
of antioxidant treatment is well supported and in some cases
effective, this kind of supplementation still presents too many
variables that require extensive analysis to conclusively prove
its effect upon the process of atherogenesis in order to be
thoroughly used in a clinical setting.
In this sense, it is interesting to mention that several years
ago, Tsimikas et al. reported levels of oxidized LDL and Lp(a)
lipoprotein in a total of 504 patients immediately before
coronary angiography was carried out . Interestingly, in the
entire group of patients studied, the association between
obstructive coronary artery disease and the ratio between
oxidized phospholipid/apoB was independent of all lipid
measurements and clinical condition except for Lp(a) lipoprotein.
With these results in hand, they were able to conclude that
circulating levels of oxidized LDL are strongly correlated in
patients who present the diagnosis of coronary artery disease
angiographically supported . Overall, this kind of clinical
data in great measure support observations and the early
proposal made by JL Witztum focusing on the role oxidized LDL
particles play during the process of atherogenesis [
Vitamins and Antioxidant Molecules
The oxidative balance of the endothelial cell is highly related to
the metabolism of lipoproteins and therefore to the development
of atheroma lesions. In this regard, patients hospitalized for an
acute myocardial infarction and, in general, populations at high
cardiovascular risk tend to present a low non-enzymatic
antioxidant capacity  and therefore a low plasma concentration of
antioxidants [29, 30]. Epidemiological evidence suggests that
plasma concentrations of different antioxidant compounds show
an inverse relationship between the seriousness of the
atherosclerotic process and its clinical manifestations, supporting the
atheroprotective properties of several antioxidants .
Carotenoids correspond to a series of compounds synthesized
by plants with redox potentials ranging from 980 to 1060 mV
 and considered as weak reductants. Interestingly,
populations showing high plasma concentrations of carotenoids,
including cryptoxanthin, lycopene, and α-carotene, present a lower
intima-media thickness than subjects with low plasma
concentrations of these compounds . In addition, α- and β-carotene
Tf-Fe(III) Rf Dasc CitC-Fe(III)
GSH F-Fe(III) CoQ Fe(III) CoQ- Asc H2O2
TxOTO C-O HPUU-FA
OC36H5ORS O2- HRP-II HOO
concentrations have shown an inverse association with
atherosclerosis when the presence of plaque in the carotid and femoral
arteries was evaluated by ultrasound . Also, when β-carotene
negatively correlates with interleukin-6, the inflammatory
process is favored . Nevertheless, despite the evidence regarding
the correlation between the protective effect of carotenoids and
the presence of a lower cardiovascular risk, statistical significance
of most results has not been maintained after correction for the
presence of risk factors such as high blood pressure and high
cholesterol levels . Similarly, no statistically
significant results were found in a population of 22,000 men
receiving for 12 years an oral administration of
βcarotene . Among carotenoids, lycopene has been
identified to be the compound with the highest reducing
capacity tested in reactions involving a singlet oxygen
(650 mV) . This reducing capacity has aroused interest
related to atherosclerosis; however, its activity has only
shown to reduce LDL oxidation in vitro , but not lipid
peroxidation or LDL oxidation in vivo .
According to ultrasonic evidence, asymptomatic patients
that show the presence of plaque in the carotid arteries in
comparison to patients with normal arteries present low
plasma concentrations of lycopene . These results are
supported by the fact that lycopene administration along with other
antioxidants drastically reduces atherosclerotic plaque in
transgenic mice models  and in healthy volunteers
improves endothelial function [41, 42]. Among these properties,
lycopene promoted the metabolism of lipids through changes
in protein expression in both in vitro and in vivo models .
Although these results suggest that lycopene presents a strong
atheroprotective capacity due to its diverse biological
activities, it is still necessary to understand the specific metabolic
pathways that become modified in order to fully explain its
molecular effects [44–46].
Since strong reductants are expected to generate better
clinical outcomes, new research employing tocopherol,
presenting a protective role for LDL oxidation, has been launched
with its natural source, vitamin E, together with several
trolox (TxOH), tocopherol (TOH), catechol (C), uric acid(UH2−),
polyunsaturated fatty acid (PUFA H2), free cysteine from protein (RS),
horseradish peroxidase (HRP) [18, 25, 26]
chemically derived molecules [23, 47–51]. In this respect,
although the presence of vitamin E in plasma shows an inverse
correlation with the development of ischemic heart disease
 and its consumption is associated with a lower incidence
of CAD , it has been observed that the molecule is
oxidized in plasma before LDL particles are affected when
synthetically oxidizing systems are employed . The
administration of α-tocopherol produced a dramatic decrease (77%) in
the risk of non-fatal myocardial infarction in a population with
a clinical history and angiographic evidence of coronary
atherosclerosis . Nevertheless, according to the α-tocopherol,
β-carotene supplementation on coronary heart disease
(ATBC) study, the molecule marginally decreased the
incidence of major coronary events and fatal coronary heart
disease (4 and 8%, respectively) . The ATBC cohort
monitored for 6 years showed that at the end of the clinical trial, the
incidence of a first-ever major coronary event non-fatal
myocardial infarction, or fatal coronary artery disease, did not
decrease. Nevertheless, the follow up of patients 2 years after
the completion of this study showed a significant decrease in
incidence rates but was not further analyzed [
other trials have shown results that were not equally
encouraging since treatment with vitamin E in a population with high
cardiovascular risk and a history of cardiovascular disease,
diabetes, and other risk factors did not reduce the prevalence
of cardiovascular-associated deaths [
] even in combination
with vitamin C, β-carotene, or both [
Ascorbic acid can be considered a reductant of average
strength with respect to the range of chemical species present
in plasma. Induced atherosclerosis in animal models, either by
promoting changes in cholesterol metabolism or by oxidative
stress, showed up to half the size of plaque in a vitamin
Cadministered group [
]. Although it is hydrophilic in
nature, ascorbic acid reduced up to 60% the oxidation of
LDL by myeloperoxidase in contrast to the lack of effects
produced by tocopherol  at plasma concentrations [
]. On the other hand, co-administration of vitamins in
experimental atherosclerosis produced superior positive changes
in the size of the atherosclerotic plaque and in the presence of
oxidation markers in comparison to treatment with each of the
antioxidants separately . Diverse clinical trials performed
in different populations presenting a high ascorbic acid plasma
concentration showed a low prevalence of angina [
Moreover, the administration of large doses was associated
with a reduced cardiovascular risk [
] in support of studies
showing that low plasma ascorbic acid concentrations are
associated with a higher incidence of myocardial infarction [
A meta-analysis of 44 clinical trials showed a relationship
between the administration of doses over 500 mg/day of
vitamin C and an improvement in endothelial function for patients
with diabetes, atherosclerosis, and heart failure, despite the
fact that no improvement was found in healthy volunteers
]. However, in a more ambitious approach, administration
of ascorbic acid to 70-year-old patients did not diminish the
incidence of deaths due to cardiovascular disease [
] nor did
modify the conventional parameters of cardiovascular risk,
such as markers for oxidative stress [
The sum of these results thus far, although not conclusive
regarding the potential benefits of antioxidant therapies, might
be related to the fact that the methodology employed is not the
same in every trial, a situation that will have to be further
investigated in the near future. Several of these studies do
not allow detecting the level of protection because specific
concentrations of antioxidants in plasma were not reported.
Additionally, several epidemiological studies lack the analysis
of the initial conditions of the total antioxidant capacity of
plasmas, since the same benefit is not necessarily provided
to all individuals with the same antioxidant dose when the
benefit due to the administration of antioxidants was recorded
only when there was a prior deficiency [
]. It is feasible to
suppose that not all people are subjected to the same oxidative
stress, that not all people have the same antioxidant defenses,
and that they all do not have the same response to the same
treatment. Since as shown in several studies these variables
have not been considered in the design and analysis of the
clinical trials, this may be one of the reasons why results range
from a 77% reduction to a lack of effectiveness when studying
the incidence of myocardial infarction. In addition, an
important premise in several of these trials has been forgotten as
oxidative stress seems to be a crucial factor during
atherogenesis and less critical when lesions are well established. Studies
employing experimental atherosclerosis show that although
the administration of antioxidants simultaneously to an
atherogenic stimulus reduces the number of atherosclerotic
lesions, in the clinic, the use of antioxidants has been mostly
limited to populations of advanced age, omitting that the
process of atherogenesis in humans may start before birth [
Due to the significant uncertainty surrounding the use of
antioxidants, treatment with stronger reductant molecules
chemically derived from tocopherol has been attempted with
promising results. The case of probucol is extremely
interesting since its antioxidant capacity has been shown to
be superior to that of the parent compound, reducing the
atherosclerotic plaque from 54 to 7% and considerably reducing
the ex vivo oxidation of LDL in animal models [
Highcardiovascular-risk populations with atherosclerotic plaque
confirmed by angiography showed a drastic reduction in
high-density lipoprotein-associated cholesterol (HDL-C), an
increase in the QTc interval, and a reduction in lumen volume
from the start of a 3-year treatment with probucol and
cholestyramine . In consideration that lumen volume is not a
clear reflection of the presence or severity of the
atherosclerotic plaque, a study was conducted in hypercholesterolemic
patients focusing on the thickness of the intima-media layer
which was reduced by 14% in 2 years under probucol
]. Nevertheless, due to the importance attributed to
maintaining the proper cholesterol metabolism during
treatment, a decrease in HDL-C was a relevant factor against the
use of probucol, which led to the search for other chemically
synthesized antioxidants that would not adversely impact the
normal lipid profile. The substitution of functional groups in
one of probucol phenolic rings yielded the synthesis of
succinobucol, an antioxidant compound that is not susceptible
to modification by metabolism, thus preventing the formation
of the hepatotoxic molecule spiroquinone and showing an
anti-inflammatory action through binding to VCAM-1 [
Although succinobucol inhibited the development of
atherosclerosis in different animal models and selectively decreased
LDL-C and increased HDL-C [
], healthy subjects treated
with succinobucol showed a decrease in HDL-C and apo AI
concentrations, changes associated with an increase in LDL-C
mainly through changes in the LDL3 subclass. These changes
were also significantly correlated with an increase in plasma
CETP mass. Moreover, administration of succinobucol to
patients subjected to a percutaneous coronary intervention did
not produce changes in the volume of plaque measured by
intravascular ultrasound compared to placebo [
patients with prior acute coronary syndromes showed a lower
incidence of myocardial infarction or cerebrovascular events
]. Interestingly, a prolonged release of succinobucol studied
in pigs using a metallic stent induced inflammation and tissue
deterioration interfering with the healing process of blood
]. These disorders may be largely due to the inhibition
of smooth muscle cell proliferation and also by an induced cell
apoptosis allowing an increment in mitochondrial ROS
through cytochrome c peroxidase activity, all of which prevent
On the other hand, the hydrophobic antioxidant BO-653
inhibited LDL oxidation and the development of
atherosclerotic lesions in different animal models compared to the effect
of probucol [
]. Rabbits with vascular damage induced by
denudation of the iliac artery under a cholesterol-enriched diet
presented an effect upon c-myc, one of the cell cycle main
controls, suggesting that the clinical use of probucol may
compromise re-endothelialization [
]. By comparison,
administration of elsibucol to rabbits fed with a cholesterol-rich
diet improved significantly IM thickness even in comparison
with probucol. Unlike succinobucol, elsibucol-inhibited
VSMC proliferation evens at concentrations four-times higher
than those found in plasma, demonstrating also a level of
reendothelialization comparable to controls. Nevertheless,
cholesterol metabolism showed some impairment since HDL-C
was decreased with no apparent effect upon LDL-C.
Although this could be considered a negative effect, it is
necessary to assess the mechanisms of modification of lipoproteins
in order to know whether this result corresponds to a retention
of particles or to a more efficient elimination of excess lipids
that could lead to a metabolic improvement [
There have been some other attempts to control
atherosclerosis with antioxidants from natural sources, like the study
from Shen et al. showing that treatment of atherosclerotic mice
with quercetin induces the expression of heme-oxygenase-1
and promotes a reduction in oxidative stress markers
attenuating endothelial dysfunction induced by a lipid-rich diet or even
strong oxidants [
]. Surprisingly, all these benefits were
obtained with a dosage close to the average daily intake
reaching a concentration level well below its detection limit
in plasma when employing highly sensitive techniques such as
HPLC coupled with mass spectrometry . Furthermore, the
administration of quercetin in LDLR−/− mice fed with an
atherogenic diet and subjected to an exercise routine showed
an enhancement of LDL resistance to oxidative modification
and a reduction of plaque [
]. Quercetin administration
associated with exercise showed an important increase in the
hepatic expression of ABCA-1, apo A-4, and PPAR-α.
However, concomitantly, there was a decrease in apo AI gene
expression, the main protein component of the widely
considered anti-atherogenic high-density lipoproteins, which
resulted in a point against a daily use of quercetin [
with the effects shown by quercetin, polyphenolic extracts
obtained from grape seeds containing compounds such as
hydroxycinnamic acid, flavonols, and stilbenes reduced
intracellular oxidative stress through a lower expression of
VCAM-1, ICAM-1, E-Selectin, MCP-1, and M-CSF that
inhibited the binding of macrophages to endothelial cells
activated by exposure to lipopolysaccharides [
]. Changes in
protein expression were primarily due to the activation of the
transcription factors NF-Κβ and AP-1 by stimulation with
lipopolysaccharides. Both factors are characteristic of systems
presenting oxidative stress associated with several
inflammatory responses and their activation [84–86].
Therefore, the statistical correlation between plasma
concentrations and the atherosclerotic status of patients
together with the existing evidence for the potential
antioxidant atheroprotective role of several molecules
represents a very encouraging scenario to search for new
molecules. We do believe it is important to highlight that natural
extracts, containing low concentrations of active molecules
and therefore difficult to measure since they are present
below their detection range, by exerting a synergistic
action might modify atherogenic patterns. This approach can
give a new direction to the search of potential antioxidant
therapies that might modify the process of atherogenesis,
not as single molecules but as a synergistically protective
Although oxidation-reduction reactions play a crucial role
in atherogenesis, analyzing antioxidants only with regard to
their reduction potential might be a too narrow perspective.
For instance, Perilla frutescens extracts and one of its
component, α-asarone, showing a strong antioxidant capacity
prevent the oxidation of LDL in vitro and in vivo . α-Asarone
potentiated the macrophage response to LXR and PPAR-γ
agonists, diminishing SR-B1 and increasing the expression
of ABCA-1 and ABCG-1 and therefore explaining its effect
through an antioxidant activity and the modulation of lipid
metabolism . Another example is sesame oil, a mixture
rich in lignans and other antioxidants that, when tested in
LDLR−/− mice fed with an atherogenic diet, significantly
inhibited atherosclerotic plaque development, mediated
partially by a favorable impact on lipid profiles . A
sesame oil aqueous extract increased the lag time in
conjugated diene formation during LDL and HDL oxidation
by exposure to Cu2+ and MPO activity . Employing
macrophages stimulated with lipopolysaccharides and
endothelial cells treated with TNF-α and moderately oxidized
LDL, the extract inhibited transcription and synthesis of
proinflammatory cytokines IL-6, Il-1a, TNF-α, chemokines,
adhesins MCP-1, and VCAM1, in addition to inhibiting
SRA1 and inducing ABCA-1 involved with cholesterol
exchange between cells and lipoproteins. This type of gene
expression is triggered by oxidative stress-sensitive transcription
factors and ligands affecting LXRs activation and NF-Κβ
inhibition and translocation . Such biochemical responses
could result from just one single multifunctional antioxidant,
or rather due to advantages from several antioxidant
compounds within the extract that might have been potentiated
Although it can be said that there is important evidence
for the potential use of antioxidants as atheroprotective
molecules in the clinic, the only way to fully support this
statement will be to importantly improve the way to
standardize their effects directly correlating their physiological
effects with their concentration in plasma. Moreover, in the
case of studying as a source for antioxidants the use of natural
extracts, the way to standardize their anti-atherogenic
effects will be only achieved by also investigating their
synergistically active components and the way these
components when present in plasma below a detection value can
provide a positive atheroprotective effect.
Regulation of Protein Expression
Compounds and molecules with a reducing ability not only
prevent physiological deterioration of vascular cells and lipid
peroxidation of LDL but can also promote or inhibit the
expression of proteins that present an atheroprotective
effect. High-density lipoproteins exhibit atheroprotective
characteristics that include mobilization of excess
cholesterol to the liver and an antioxidant activity associated with
paraoxonase-1 (PON1) [92–94]. PON1 hydrolyzes lipid
oxidation products, prevents their formation reducing
intracellular stress of macrophages in vivo , and presents
a reduction capacity that has been shown to be diminished
in plasma from patients with a recent myocardial infarction
and those at high cardiovascular risk . Another
characteristic of this enzyme involves the improvement of
cholesterol efflux by macrophage ABCA-1 expression
promoting binding through its amphipathic helices with
membrane cholesterol lipid rafts  and a catalytic core
composed of glutamic acid, asparagine, and aspartic acid with
activity dependent on free thiols [97, 98].
On the other hand, it has been described that after
delipidation, HDL particles show an antioxidant capacity
independent from PON1 . In this respect, Kotosai et al.
showed that apo AI reacts selectively with free fatty acid
hydroperoxides in a mechanism dependent on methionines,
reducing these compounds to stable oxidation products such as
fatty acid hydroxyls . Other apolipoproteins associated
preferentially with HDL effectively bind oxidized LDL
phospholipids, reducing their rate of oxidation and increasing the
lag time for the process to take place . In general, cellular
responses to modified protein structures could be an essential
step to understand pathogenesis and therefore the atherogenic
Since proteins account for approximately 70% of the dry
cell mass, assays conducted on cells under constant generation
of hydroxyl radicals (E°′ = 2310 mV) show protein peroxides
as the main oxidized product with almost nonexistent lipid
and nucleic acids oxidation products  and where the
generation of protein peroxyl radicals weakens intracellular
antioxidant defenses . In this regard, the efficient defense
system shown by ascorbic acid seems to reside in both
preventing the formation of peroxyl radicals and the
acceleration of the decay process of these radicals to stable hydroxide
species [105, 106]. Ascorbate also protects the intracellular
levels of glutathione, where both antioxidants combined
prevent further intracellular protein peroxide formation .
Considering glutathione as a strong reductant (E°′ = −
1500 mV), it is to be expected that its presence in plasma
attenuates early vascular lesion development as observed in
a hyperlipidemic mice model [108, 109]. It has been observed
that glutathione plasma concentration seems to be a critical
factor in the development of the atherosclerotic plaque since
an 80% decrease in its intracellular concentration promotes
the development of complex vascular lesions [103, 109].
This information is further supported by experiments where
bone marrow transplantation in experimental animals capable
of synthesizing up to three-times more glutathione than
normal, reduced the progression rate of vascular lesions
by approximately 35% .
Several models for atherosclerosis such as transgenic mice
with humanized and pro-atherogenic lipid metabolism have
shown by the administration of ribose-cysteine an increased
glutathione and glutathione peroxidase activity in liver tissue
and plasma. This modification appears to be responsible for a
reduced content of oxidized biomolecules in the liver, plasma,
and aorta, in addition to a reduction in LDL-C, apoB, Lp(a),
and total cholesterol plasma concentration associated with an
increase in LDLR expression .
Both the concentration and activity of proteins are altered
through oxidative modification, regulation of expression, and
post-transcriptional silencing of non-coding RNA fragments
(micro RNA or miRNA) paired to the 3′ untranslated regions
from target genes .The human genome encodes
approximately 1800 miRNAs capable of regulating protein function
through direct binding to their 3′UTR  and by several
indirect mechanisms through regulation of repressors or
transcription factors . Therefore, miRNAs have proven to
impact the development of atherosclerosis, for instance,
inhibiting LDLR and ABCA1 expression in hepatic cells
. Overexpression of miRNA-223 down-regulated SRBI
and HMGCS limiting cholesterol synthesis and indirectly
increasing ABCA1 due to the modulation of transcription factor
Sp1 . In hepatocyte cell cultures, miRNA-27a decreased
by 40% the levels of LDLR increasing the concentration of
PCSK9, which enhances LDLR degradation and regulates
LRP6 and LDLRAP1 . Results from in vivo models
provide an even more exciting scenario since stable
atherosclerotic lesions show different patterns for miRNAs in
comparison to complex lesions prone to rupture, where
inhibition of miRNA-494 led to a decrease of plaque size
and the presence of more stable lesions .
However, the inhibition of specific miRNAs in cell culture
cannot be easily extrapolated to a therapeutic approach since
the presence of miRNAs in circulation might affect not only
the vascular tissue but also other organs and cell systems while
being transported by HDL [116, 117]. Proteins such as PON1
with intrinsic antioxidant capacity are also regulated by
specific miRNAs . On the other hand, several antioxidant
compounds such as resveratrol modulate the expression
patterns of several miRNAs involved in both cancerogenic and
inflammatory processes . A clinical trial involving
male patients suffering from hypertension with associated
diabetes mellitus type2 and coronary artery disease, after
1 year of an 8-mg daily intake of resveratrol, revealed a
decrease in the expression of proinflammatory cytokines
modifying the level of several miRNAs . Orally
administrated polyphenols to hyperlipidemic mice also prevents fatty
liver disease through the action of miRNA103 and
], whereas catechins are capable of changing
the expression of hepatic miRNAs in apoE-deficient mice and
HepG2 cells [
All this evidence lead us to further evaluate the potential
effects of miRNAs as key regulators of many antioxidant
proteins involved in the defense mechanisms against ROS
generation. Among all miRNAs that might be modulated by
antioxidant compounds, we need to keep in mind that exogenous
miRNAs can be absorbed from the diet, and therefore, found
in plasma where they could also modify gene expression
]. Zhang et al. have proven that miRNAs derived from
plants can be absorbed in the gastrointestinal tract reaching the
plasma in stable microvesicles showing an effect upon
Perspectives for Antioxidant Therapy
To date, accumulated evidence regarding oxidation as a
proatherogenic factor indicates that biochemical events involved
in this process are indeed a very attractive target for the
management of cardiovascular disease in the clinic.
Nevertheless, although evidence indicates that redox
reactions are important in the initiation and progression of
atherosclerosis, oxidation with a pro-atherogenic context does
not eliminate the fact that oxidation participates in many
cases as an essential messenger of cellular signaling
]. Therefore, disease management and therapeutic
goals require not only high-precision and high-sensitivity
methods to detect in plasma very low amounts of reducing
and oxidizing molecules but also a much better
understanding of the normal processes and metabolic pathways
influenced and/or controlled by oxidative stress. As several
methodologies have been specifically described for the
quantitation of the total antioxidant capacity and the oxidation state
of diverse biological systems [25, 26], a successful way to
carefully study how redox reactions influence atherosclerosis
can be achieved. Nevertheless, since there is still a lack of
standardization in many of these methods, clinical trials
studying antioxidant capacity have been difficult to compare
and therefore difficult to use in order to reach a conclusion.
On the other hand, there is also a problem to consider
related to the possibility that in many of the studies discussed in
Fig. 2 Interaction between ROS and antioxidants. Since LDL particles
present in the subendothelial space constantly remain exposed to ROS,
lipoproteins gradually become transformed into oxidized particles
(oxLDL) (1). Cells exposed to oxLDL activate transcriptional factors
and several receptor expression patterns leading to cell metabolism
changes (2). Phagocytosis of oxLDL carried out by macrophages
promotes their transformation into foam cells prone to release
intercellular signaling molecules that favor inflammation (3).
Nevertheless, the presence of antioxidant molecules in the
subendothelial space may avoid LDL oxidation (4). Antioxidants react
with reactive oxygen species (ROS) (5), exerting a protective role against
cellular damage due to oxLDL formation (6). Antioxidants might
enhance or diminish the presence of specific receptors and intracellular
enzymes that in general promote the presence of an anti-atherogenic
metabolic status (7). Despite oxidation, interaction with HDL can
regenerate LDL from oxLDL (8)
this review, the concentration of the specific antioxidant did
not reach the critical active concentration at key sites known
to be important in the development of atherosclerosis, for
instance the intima of blood vessels or the hepatocyte. This
is an important point that will have to be technically solved
in order to have the certainty that future clinical trials will
present the possibility to directly correlate specific tissue
concentration of antioxidants and prevention with the
development of the disease.
Since we have recently found a direct relationship
between the exposure of not only chemically modified LDL
but also normal LDL going through a “normal” process of
oxidation with specific transcriptomic changes in vascular
smooth muscle cells [
], several antioxidant molecules
support their activity by creating a proteomic or even a
miRNA pattern that leads cell expression to what can be
recognized as an antioxidant metabolism pattern (Fig. 2).
Nowadays, a broad spectrum of tools including
proteomic, metabolomic, and transcriptomic approaches have
been developed to render detailed information related to
the many changes found in the metabolism of cells due to
a specific antioxidant treatment [
challenging research providing brand new data and more
importantly brand new concepts is ahead of us. For
instance, to find an efficient way to target antioxidants to
specific intracellular organelles such as lysosomes and
mitochondria, or to study subtle changes that might occur
in the epigenetic control of gene expression secondary to
antioxidant therapy, could be considered as two
interesting approaches. We believe a comprehensive analysis of
this new knowledge and its relationship with the presence
of plasma antioxidants and their reducing capacity will
undoubtedly open new ways to understand and develop
new therapeutic pathways in the fight not only against
atherosclerosis but also against other degenerative
Compliance with Ethical Standards
Conflict of Interest Paola Toledo-Ibelles and Jaime Mas-Oliva declare
no conflict of interest. Studies by J.M-O research group described in this
review were supported by CONACYT (Grants 180726 and 255778) and
DGAPA-UNAM (Grant IN-205814-3). P.T-I received a scholarship from
CONACYT during her graduate studies (Posgrado en Ciencias
Bioquímicas, Universidad Nacional Autónoma de México).
Human and Animal Rights and Informed Consent This article does not
contain any studies with human or animal subjects performed by any of
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